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Application of Molecular Techniques to Improved Detection of Insecticide Resistance Kathleen K. Brewer T he significance and extent of resistance to insect and acarid control agents is generally appreciated as a major obstacle to pest control, as well as an environmental concern. Insecticide resistance is in transition from examination of gene products to the genes and their expression. Several gross chromosomal alterations already have been detected. Further details of these, as well as the molecular basis of more subtle adaptations, can be expected to be revealed shortly and validated in field-selected, economically significant pests. There is precedence for this optimism. Localization and elucidation of nucleotide sequences have been quickly followed by the development of DNA assays for TaySachs disease, hemoglobinopathies, and Lesch-Nyhan syndrome, to name only a few. The human anomalies cited above are well-characterized biochemically and represent messenger-RNA-abundant proteins, like many instances of metabolic insecticide resistance. The availability of antibodies and related sequence data from other species will help with the initial obstacle, which is localization of resistance genes. The ability to perform crossing experiments with insects, as well as short generation times and favorable polytene chromosomes in dipteran pests, also is beneficial. Technical advances also suggest that many resistance mutations may be revealed shortly. For example, newer vectors and techniques to sequence DNA directly have accelerated mutation research by bypassing tedious, and more risky, cloning steps in cases where the wild-type sequence is known. These observations, as well as the recent explosion of interest in this area, suggest that it is not premature to think about ways in which investigations of the molecular basis of insecticide resistance might contribute to pest management. 96 AMERICAN ENTOMOLOGIST Various methods are available to estimate the frequency of resistant phenotypes. Field trials, dose response, and discriminating dose bioassays initially are useful for documenting the presence of adaptation or actual resistance and for estimating the efficacy of control agents. Several authors have indicated that more specific and sensitive methods are needed for estimating the frequency of resistance mutations in both pests and beneficial insects before control failures occur, as well as for evaluation of management tactics. A related topic, plant-insect interactions and host-race formation, also would gain from more sensitive means to detect genetic heterogeneity. Molecular techniques already have been applied to herbicide, antibiotic, and antineoplastic resistance, and these applications could provide useful paradigms for entomology. Here, requirements for resistance assays are summarized, gaps in detection of insecticide resistance identified, followed by discussion of potentially useful molecular tools. These techniques undoubtedly are wellknown to molecular biologists, but may be unfamiliar to other entomologists. Here, the emphasis is on resistance to synthetic insecticides. However, the natural variation in sensitivity and evolution of resistance to Bacillus thuringiensis in field and artificial situations, for example, suggest that a means to detect genetic heterogeneity to this and other biopesticides also is needed. he ideal assay for detecting resistance is sensitive enough to test single insects, specific for a resistance factor, and capable of resolving heterozygous and homozygous genotypes. The assay should be adaptable to screening large numbers of insects and should be able to provide information regarding the selectivity of control treatments. The assay also should identify a discriminating dose, and the system should permit more than one test per specimen. The ultimate goal includes the development of cost-effective and widely accessible assays that can be implemented in the field. In addition, a means to simultaneously detect the presence of pathogens is desirable for disease vectors. Assays with many of these features have been developed for esterases, glutathione-S-transferases (GSTs), and resistant forms of acetylcholinesterase. T Requirements Microassays T Gaps in Detection Resistance here are instances where microassays for metabolic resistant factors have not been forthcoming or are incomplete. (The following summary provides little in the way of new information or syntheses but I've included it here to put the potential of molecular tools in perspective.) The development of single insect assays for the mixed function oxidases (MFOs) has been complicated by problems with extraction, stability, purification, and overlapping substrate preferences of isoenzymic forms. The GSTs also have a pattern of overlapping catalytic capability without real specificity. Measurement of general esterase activity with a surrogate substrate may not distinguish between variants with enhanced titers versus mutants with altered specific activity. Assays that test whole enzyme titers can be misleading, because only one isozyme actually may be involved in resistance. Also, there are cases of metabolic resistance where additional information regarding the genotype is essential. For example, Devonshire and his colleagues have combined an immunoassay with a DNA probe to identify resistant genotypes in Myzus persicae (Sulzer). This has practical consequences because revertants constitute obscured resistance potential within the population. There is a parallel to this in prenatal diagnosis of a lipid storage disorder, in which the enzyme assay cannot resolve intractable infantile and less severe adult phenotypes; molecular analysis is required to make this distinction. There are also adaptations of the nerve insensitivity type that may require DNA probes. Alterations in the expression of sodium channel protein and the phospholipid content of membranes have been correlated with nerve insensitivity. These might be assayed with a quantitative immunological assay or biochemically, respectively. But nerve insensitivity also is believed to include structural mutations in target macromolecules-the sodium channel and gamma-aminobutyric-acidchloride ionophore complex. Such structural mutations could be refractory to Summer 1991 for Resistance of Insecticide 97 immunological assays because they are likely to be which may be lost with solubilization. So there are been refractory to the development of microassays, from assays directed toward the gene, or in addition Molecular Techniques with the Potential for Application to Detection of Insecticide Resistance changes in the configuration, cases of resistance that have and others that might benefit to the gene product. T here are both indirect and direct methods for detection of mutations that might have application to integrated pest-management (IPM). (The descriptions provided here are intended for orientation only.) Linkage analysis is an indirect tool that relies on markers coinherited with a mutation at a statistically reliable frequency. Markers can be phenotypic traits such as eye color, but DNA markers, known as restriction fragment length polymorph isms (RFLPs),are used more often. The molecular basis of RFLPs are benign single-base alterations scattered throughout the population. These polymorphisms are sensitive to digestion with restriction enzymes (fig. 1). Restriction enzymes are bacterial-derived endonucleases that usually recognize and cleave (A) palindromic sequences-that is, shorr stretches of nucleotides that read the same in both directions, as in the example below for Hpa II. (5')CC-G-G(3') (3')G-G-CC(5') Patterns of restriction fragments are visualized by electrophoresis (fig. 2). Linkage analysis can be invaluable for screening when the gene has not been isolated, as in Huntington's disease, or when many mutations cause a similar phenotype. Fig. 1. Two means of detecting mutations. Restriction fragment analysis (above). Nucleotide changes can be associated with restriction enzyme site cleavage. This is useful when the change is benign (polymorphism) but is coinherited with a deleterious phenotype. Restriction fragment analysis also can be used when a mutation itself changes the pattern of restriction enzyme digestion. Alternatively, (below) mutations can be directly detected by hybridizing with a nucleotide probe to the normal sequence. Because the mutation causes a mismatch, it is unstable and will be washed off, in contrast to normal. Restriction fragment analysis ~-_•... ~-.-----~ ~ Allele-specific oligonucleotide hybridization ,• 98 Target nucleic acid Hybridization probe Point mutation Site of enzyme action AMERICAN ENTOMOLOGIST ~ CLEAVE Fig. 2. Traditional means of detecting RFLPs. Total (genomic) DNA is cut with restriction enzymes and separated by electrophoresis. DNA is then transferred to an immobilizing filter. A probe with some type of intensifying signal is added. The unbound probe is washed off following an incubation period. Patterns of RFLPs are revealed following autoradiography. The polymerase chain reaction can eliminate the need for the hybridization step, since amplified and digested fragments are visible following electrophoresis. DNA ~--- WITH RESTRICTION -\ AGAROSE ENZYMES / GEL ELECTROPHORESIS ---'\ '---TRANSFER TO NITROCELLULOSE GEL NITROCELLULOSE 1/1 HYBRIDIZE WITH LABELED PROBE •• WASH 1/2 2/2 Alleles (kb) ~3.0 L=.J 2.0 RFLPs Probe •• •• Allele 1 3.0 kb Allele 2 Alternatively, mutations may be revealed directly by hybridization studies, the essence of which is the difference in binding stability between a chain of nucleotides, referred to as a probe, containing the wild-type sequence and a corresponding stretch of mismatched DNA or RNA (fig. 1). Samples of DNA bound to a solid support are incubated with a probe that corresponds to the sequence of interest. When washed under conditions of correct stringency, only probes that match perfectly remain. Two conditions must be met for this to work. First, the probe must find its target sequence. A larger nucleotide probe can find its complementary sequence in the vast amount of DNA that constitutes a genome more easily than a smaller probe, or when nature has provided an increased copy number of the target sequence. Second, washing conditions must be such that only perfectly matched probes remain. As a general statement, gross chromosome rearrangements, deletions, or insertions are detected most readily as described here. Gene amplification has been shown to be the molecular basis of two cases of resistance to date, and quantitative hybridization assays are available for these. Many as yet undiscovered resistance mutations are believed to be single nucleotide substitutions, and these subtle adaptations require more sensitive means to Summcr I f.)f.) I 99 detect them. Formerly, probes of at least 100 nucleotides in length were required to find their corresponding sequences in total (genomic) DNA. But probes of this size have small differences in thermal stability between a perfectly matched hybrid and one containing a single mismatched base pair. Consequently, single nucleotide substitutions usually could not be detected with probes of this size unless they fortuitously abolished or created a restriction enzyme site, as in sickle-cell anemia. But palindromic sequences are represented by approximately one in every six nuc1eotides. For example, only about half of human disease mutations are detectable by this method, illustrating the considerable limitation to this approach. The laborintensive, multistep, and capricious nature of conventional hybridization analysis, as well as slow turnover time, limited applications of this technique. Within· the past few years, polymerase chain reaction (PCR) technology has provided a means to increase the copy number of any targeted sequence. This has increased the sensitivity and accessibility of hybridization studies in general. Detection of point mutations is much easier following PCR. Shorter probes (seventeen to twenty-one nucleotides) can find their targets more readily, since there are a billionfold copies. In addition, shorter probes are more sensitive because they are readily washed off if a single mismatch (mutation) is present (fig. 1). The PCR is an in vitro amplification scheme consisting of a series of incubation steps at various temperatures, starting with extremely small amounts of DNA for the initial substrate. Each cycle consists of the following steps (fig. 3): 1. DNA is heat denatured to single-stranded templates. 2. Flanking nucleotide primers are annealed to their corresponding sequences at a low temperature. These primers are short nucleotide chains that do just what the term implies-they are obligate starting points for the polymerase enzyme. 3. The thermostable polymerase fills in corresponding nucleotides at a higher temperature, using the single-stranded DNA as a template and four nucleotide building blocks. Thus, each strand is reproduced with each cycle and is a template for the subsequent reaction, resulting in an exponential increase in copy number of a desired nucleotide sequence within hours. The results of this reaction can be easily visualized by electrophoresis in the presence of ethidium bromide, a dye that is selective for nucleic acids. Several applications of the PCR have potential for improved detection of insecticide resistance. RFLPs or mutations that cause the loss or gain of a restriction site may be studied more easily with this method because amplified and digested DNA can be visualized without hybridization. When hybridization is required, nucleotide probes to normal and mutant sequences are selected. They are long enough to represent unique sequences in the genome, yet short enough to be destabilized by a single internal mismatch. There are instances where metabolic resistance factors might be studied in messenger RNA (mRNA), for which the PCR also is applicable. This is done by making complementary DNA (cDNA) from an RNA template using reverse transcriptase. This enzyme (reverse transcriptase) is so called because it works in the opposite direction from normal transcription, making DNA from RNA. cDNA is that portion of a gene that an organism actually uses; in other words, noncoding, extra genetic material is not included. In a second reaction, PCR is performed as already described. This two-step process is less complicated than it sounds. mRNA does not have to be isolated from other RNA species, and both reactions can be done in the same buffer system. Metabolic resistance variants with more enzyme protein versus mutants with more active enzyme species might be resolved in this manner. This approach has been used to screen for thymidylate synthase in cisplatin-resistant tumors. It might be necessary also to look at mRNA when the proteins of interest are difficult to extract from single insects, as for the MFOs. Additional applications of the PCR also have potential for IPM. Because this technique permits detection of minute samples of DNA, infectious agents-those of public health interest, as well as plant pathogens-can be detected in pest populations, as has been shown for the Lyme disease spirochete. The tools of 100 AMERICAN ENTOMOLOGIST original DNA peR primer new DNA DNA + primers + dNTPs + DNA polymerase + ••I denature and synthesize i i I Fig. 3. Polymerase chain reaction. DNA is separated (denatured) by heating. Probes specific for the target sequence prime the synthesis of new DNA in the presence of nucleotide building blocks (dNTPs) and DNA polymerase, a DNA replicating enzyme. Because each new strand of DNA is a template for the subsequent reaction, sequential cycles cause exponential amplification, resulting in greater than a millionfold increase of a target fragment more than twenty to forty cycles. ! I iI I t + , denature and synthesize , ! t ! ;. + ••I I ! Ii ! •• ••j I ;. +i I I i I • ! I I It I ii ,I I + denature and synthesize II Ii t! Ii I : It I !I i i I..i • I : t i ~I i I ~i ;. + ••i I • I I I I I I • ~i continue for 20-40 cycles linkage analysis and hybridization are hardly new, but more recent techniques have made them potentially useful tools for IPM. T he nucleic acid approaches outlined above are specific and sensitive enough to assay single, even minute, insects. Direct detection using either probes or restriction enzyme digestion has excellent resolution because wild-type, heterozygous, and homozygous genotypes can be distinguished. Detoxifying enzymes can vary with developmental stage and diet-a possible variable in sampling for metabolic studies-but not for DNA assays. It appears likely that multiplex screening of each specimen, either metabolic, immunological, or genetic, would be compatible with DNA diagnostics. Simultaneous detection of pathogenic organisms in disease vectors and monitoring of insect pathogens and parasites, either by immunological or molecular means, seems feasible too. Also on the positive side, the peR is a well-established technique, which is accessible to investigators without previous experience in molecular biology. The reaction is relatively robust, once conditions are optimized. Preliminary experiments generally involve finding the most efficient sets of primers and determination of the optimal annealing Summer 1991 Anticipated Advantages and Limitations to Detection of Insecticide Resistance Using Molecular Techniques 101 temperature. However, there will be biological, statistical, and technical obstacles and limitations to the application of DNA diagnostics to IPM. The major caveat in interpretation of DNA diagnostics is that they test only the genotype, unlike the best assays, which include the feature of testing response to control agents. Primary discriminating dose assays provide response data. In addition, DNA diagnostics are limited by their specificity; more than one mutation might confer the same resistant phenotype. However, most cases of resistance are believed to be caused by a single mutation within a population. Sessile and parthenogenetic insects could be notable exceptions, because they actually may be series of isolated demes within populations. Estimations of polygenic resistance may be inflated by studies with laboratory-selected insects, because there are good reasons to believe that such experiments tend to select for multifactorial resistance. Linkage analysis may prove to be somewhat dynamic with respect to specificity, because the coinheritance of genes can drift within a population over time. Note also that restriction enzymes may fail to cut if any of the nucleotides in the recognition sequence or cleavage site are altered; consequently, analysis by this method is slightly less precise. Limitations because of specificity may vary with the biology of the pest, the selection conditions, and the assay. Insecticide resistance is essentially a population phenomenon. This is a crucial point in adapting DNA diagnostics to IPM. It is believed that several hundred insects are requjred to detect resistance mutations before control failures occur. But the number of samples required for molecular assays will be a subset of the initial bioassay. DNA or RNA sample preparation will be dictated by the application. Sometimes cell lysates are sufficient preparation for molecular studies. Merely squashing insects on a nylon filter can be adequate for hybridization studies if multiple copies of the target sequence are already present. The PCR requires varying degrees of nucleic acid purification. Because of the amplification potential of this technique, only a small amount of starting DNA is required, so inhibitors are diluted. In addition, the initial heat denaturation step eliminates many other troublesome agents. Simple cell lysates and a nuclease inhibitor often suffice. This is not the case when the target is an infectious agent present in only a small proportion of cells, or when the PCR is inefficient, as paradoxically occurs for some sets of primers. In these instances, more target DNA is needed and therefore, more rigorous purification is indicated. Restriction enzymes also are sensitive to contaminants and indeed may have anomalous activity in their presence. Complete DNA or RNA purification involves lysis of cells, inhibition of endogenous nucleases, degradation of membrane components, and pelleting of the nuclear fraction. Because mRNA is in the cytoplasm, this fraction is saved for RNA extraction. Proteins are removed by phenol-chloroform extractions, followed by selective ethanol-salt precipitation of nucleic acids. Because of the tissue-specific nature of many mRNAs that are relevant to resistance and the ubiquitous and hardy nature of endogenous RNA-degrading enzymes, purified RNA often may be required. These RNA protocols probably will have to be individualized, because methods to isolate RNA do not work equally well with tissues from various sources. Sample preparation for molecular assays, then, will vary from simple to rigorous. While the power of the PCR is partially because of its amplification potential, this also raises some unique considerations for preparation of DNA and RNA from insects. Fertilized gametes will have to be excluded or accounted for. Homologous or partially homologous sequences in microbial symbionts also could confound quantitative studies or introduce PCR artifacts. Experimental controls for cross contamination between samples and reagents always are necessary. The PCR reaction and subsequent steps are not particularly labor intensive. A single worker can process approximately 120 samples per day using an automated thermal cycler. A mini-electrophoresis apparatus, similar to that used for screening monoclonal antibodies, is useful for examination of many peR products. Dot blot formats allow hybridization of many samples simultaneously. When probes are required, a means to visualize them after hybridization is necessary. This is done by replacing the terminal phosphate of short probes with some type of reporter 102 AMERICAN ENTOMOLOGIST molecule, usually a radioactive isotope. But chromogenic nonradioactive reporter schemes that are permanent and less hazardous also are available. Signal intensity with both of these types of reporters usually is enough to permit overnight development. The greatest bottleneck in application of molecular techniques to detection of insecticide resistance is likely to be the amount of sample preparation required. There are universal technical considerations as well. The nucleotide sequence of at least one single copy region adjacent to, or including, the mutation or RFLP must be available so that complementary nucleotide primers can be synthesized. The size of the genome must be estimated for each pest so that primers and probes that are likely to hybridize to unique sequences can be designed. Also on the debit side, it is impossible to imagine how nucleic acid assays could be conducted in the field. But this limitation applies to some of the metabolic and immunological assays as well, and it does not preclude their use. While there are expected benefits from the application of molecular techniques to rPM, there also will be limitations and practical problems to be resolved. ome molecular assays can be expected to have more widespread application than others. Metabolic resistance adaptations may be either quantitative or qualitative. All manner of favorable mutations seem possible. Deleterious mutations in the well-studied human beta-globin gene, for example, provide a reverse analogy for how heterogeneous the molecular basis of metabolic resistance might be. As previously mentioned, the number of resistance mutations within interbreeding populations is thought to be limited but could be variable between reproductively isolated populations. Linkage analysis can be most useful in situations like this when it is not possible or not cost-effective to screen for every mutation. Some nerve insensitivity adaptations are likely to be structural mutations, and these are expected to be relatively homogeneous at the molecular level because of requirements for normal signal transmission by the target macromolecules. Such nerve insensitivity factors may be the best candidates for direct assays because a more limited battery of nucleotide probes would be required. Nucleic acid assays will not supplant other procedures. The limitations of diagnosis by genotype indicate that this approach should be confined to cases of resistance that do not lend themselves to assays for gene products or that would benefit from a dual phenotypic and genotypic assay. Indirect tools such as linkage analysis will be most useful initially; some will be replaced later by direct tests. Where insect collection or extensive sample preparation is impractical, molecular assays may have utility only in laboratory simulation studies. Less is known about variation in susceptibility to biological control agents and genetically engineered crops. It is hoped that all available technology will be recruited for detection of genetic heterogeneity to these newer agents as well. 0 S Expected Contribution of Molecular Techniques to Insecticide Resistance Management L. B. Brattsten, A. L. Devonshire, R. T. Roush, R. M. Sawicki, and D. M. Soderlund generously provided reprints or galley proofs of manuscripts in press. C. W. Holyoke, Jr., and anonymous reviewers made very helpful comments on earlier versions of this manuscript. Acknowledgment Kathleen. Brewer received her masters of science degree in entomology from the University of Delaware. She is completing predoctoral studies at Thomas jefferson University. Her research problem deals with the molecular basis of a Tay-Sachs disease variant. Summer 1991 103